Advanced molecular diagnostic techniques for detection of food-borne pathogens: Current applications and future challenges.

The elimination of disease-causing microbes from the food supply is a primary goal and this review deals with the overall techniques available for detection of food-borne pathogens. Now-a-days conventional methods are replaced by advanced methods like Biosensors, Nucleic Acid-based Tests (NAT), and different PCR-based techniques used in molecular biology to identify specific pathogens. Bacillus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Campylobacter, Listeria monocytogenes, Salmonella spp., Aspergillus spp., Fusarium spp., Penicillium spp., and pathogens are detected in contaminated food items that cause always diseases in human in any one or the other way. Identification of food-borne pathogens in a short period of time is still a challenge to the scientific field in general and food technology in particular. The low level of food contamination by major pathogens requires specific sensitive detection platforms and the present area of hot research looking forward to new nanomolecular techniques for nanomaterials, make them suitable for the development of assays with high sensitivity, response time, and portability. With the sound of these, we attempt to highlight a comprehensive overview about food-borne pathogen detection by rapid, sensitive, accurate, and cost affordable in situ analytical methods from conventional methods to recent molecular approaches for advanced food and microbiology research.

Evidences for diabetes and insulin mimetic activity of medicinal plants: Present status and future prospects.

Diabetes mellitus (DM) is a considerable systemic metabolic disorder to exhibit various metabolic and cardiovascular disorders, mainly hyperglycemia. The global projected estimate of diabetes in 2030 will be about 439 million adults, out of which 300 million expected are of type-2 diabetes mellitus (T2DM). The present knowledge revealed responsible factors, occurrence and mechanism of these factors involved in the DM diseases. Hence, the aim of this review is to address and summarize the causes, plant resources, importance, present status and future programmes for diabetes control. The present review answers the contemporary present questions raised in the scientific field on DM. Two major problems are explained in detail about the autoimmune attack or dysfunction of ß-cell and insulin resistance involved for Type 1 and Type 2 DM, respectively. Though there are various approaches to reduce the ill effects of diabetes and its secondary complications, many preferred herbal formulations due to lesser side effects and low cost. For this reason still it is getting increased attention in searching antidiabetic medicinal plants for hot research and to develop targeted medicine. Recurrence of islet autoimmunity lesson from pancreatic islet cell transplantation to cure T1D was outlined. With these highlights, the review summarizes the current knowledge on diabetes occurrence, factors (environmental and genetics), and types (I, II, gestation, and secondary DM), antidiabetic plants, sources for insulin mimetic plant principle compounds and their target mechanism with current and future trusted research areas for controlling of DM.

Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum.

Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

Green synthesis of silver nanoparticles by Rivina humilis leaf extract to tackle growth of Brucella species and other perilous pathogens.

Novel approaches are obligatory to treat chronic intracellular bacterial infectious diseases like Brucellosis specifically, are very complicated to deal with. The aim of the study to take upon nanotechnology approach to exploit the efficacy of the synthesized nanoparticles, to overcome barriers for treatment of Brucella species and other pathogens. Present study used Rivina humilis extract as reductant of silver ions for synthesis of silver nanoparticles for the first time. Rh-AgNP's was characterized by UV-visible spectroscopy, DLS, FT-IR, SEM, EDS, TEM and XRD. Radical scavenging, antibrucellosis, bactericidal activity was evaluated. Clinical application was assessed by Rate of haemolysis, fibrinolytic and Hemagglutination activity. UV-visible spectrum of synthesized Rh-AgNP's showed maximum peak at 440 nm indicating the formation of nanoparticles. TEM showed that the average particle size of nanoparticles 51 nm with spherical shape, DLS depicted monodisperse state in water; EDS confirmed the presence of silver metal. Rh-AgNP's exhibited potential antibrucellosis activity against B. abortus, B. melitensis and B. suis effective inhibition at 800 µg/mL. The bio-compatibility of Rh-AgNP's was established by rate of haemolysis, hemagglutination and fibrinolytic activity. For the first time it has been proved that Rh-AgNP's have efficacy as antimicrobial agent with potential application in the biological domain.

Synthesis and antimicrobial activity of 1-benzhydryl-sulfonyl-4-(3-(piperidin-4-yl) propyl)piperidine derivatives against pathogens of Lycopersicon esculentum: a structure-activity evaluation study.

Several 1-benzhydryl-sulfonyl-4-(3-(piperidin-4-yl)propyl)piperidine derivatives 8(a-j) were prepared by the treatment of substituted benzhydryl chlorides with 4-(3-(piperidin-4-yl)propyl)piperidine followed by N-sulfonation with sulfonyl chlorides in the presence of dry methylene dichloride and triethyl amine. The synthesized compounds were characterized by (1)H-NMR, IR, and elemental analysis. All the synthesized compounds were evaluated in vitro for their efficacy as antimicrobial agents by artificial inoculation technique against standard strains of two important bacterial viz., Xanthomonas axonopodis pv. vesicatoria and Ralstonia solanacearum as well as and two fungal pathogens namely Alternaria solani and Fusarium solani of tomato plants. We have briefly investigated the structure-activity relation studies and reveal that the nature of substitutions on benzhydryl ring and sulfonamide ring influences the antibacterial activity. Among the synthesized new compounds 8b, 8d, 8g, 8h, 8i, and 8j were showed significant potent antimicrobial activities compared to the standard drugs chloramphenicol, mancozeb.

A novel indicator plant to test the hypersensitivity of phytopathogenic bacteria.

Hypersensitive response is an important, definitive test to separate plant pathogenic bacteria from saprophytes. The current standard protocol uses tobacco and four o'clock plants as indicators for Gram negative and Gram positive phytopathogenic bacteria, respectively, and inoculation is accomplished by infiltrating bacterial suspensions into intact leaves. Both plants, especially the four o'clock, have thin leaves which make inoculation difficult, sometimes leading to inaccurate tests. Here we propose the use of Sedum hybridum as an alternative indicator plant. Sedum plants are readily available, easy to propagate and fast growing. Their leaves are much thicker, thus infiltration of a bacterial suspension is easier compared to tobacco and four o'clock. Additionally, sedum plants can be used universally to test both Gram negative and Gram positive phytopathogenic bacteria.

Synthesis of novel benzodioxane midst piperazine moiety decorated chitosan silver nanoparticle against biohazard pathogens and as potential anti-inflammatory candidate: A molecular docking studies.

Nanoparticles (NPs) are currently being investigated along with the use of biodegradable polymer containing active agents in many areas of medicine for targeted applications. The present study was aimed to synthesize novel compound Benzodioxane midst piperazine (BP) and characterization of a BP decorated chitosan silver nanoparticles (BP*C@AgNPs) and shown effective against hazardous pathogens, and also having anti-inflammatory property. It was further evaluated for molecular docking proofs, and toxicity. The BP*C@AgNPs had spherical shape with size of 36.6nm with wide biocidal activity against hazardous Gram-positive and Gram-negative bacteria with excellent inhibition at 100µg/mL for S. aureus (10.08±0.05mm ZOI), and E. coli (10.03±0.04mm ZOI) compared to antibiotic Streptomycin. The anti-inflammatory activity exhibited IC50 value of 71.61±1.05µg/mL for BP*C@AgNPs compared to indomethacin (IC50=40.15±1.21µg/mL). Also, the docking study of BP showed excellent score for COX1 and DNA gyrase. This in silico study confirmed the achieved efficacy of BP, with less toxicity against normal PMBCs in vitro and in vivo studies. This study concludes that, the novel synthesized BP*C@AgNPs had excellent biocidal property and as anti-inflammatory candidate revealed by docking studies, it confirms BP*C@AgNPs for first-class therapeutic applications in the area of medicinal nanotechnology for the coming days.

Photocrosslinker technology: An antimicrobial efficacy of cinnamaldehyde cross-linked low-density polyethylene (Cin-C-LDPE) as a novel food wrapper.

In recent years much attention has been devoted to active packaging technologies that offer new opportunities for the food industry and food preservation. The spoilage of food products during post process handling leads to food contamination and causes life-threatening food-borne illness. The methicillin-resistant Staphylococcus aureus (MRSA) 090 is one of the food-borne pathogens associated with food poisoning that leads to an outbreak of perilous human infections worldwide. The development of resistance in bacteria and diffusion of coated synthetic preservatives into food are the major problem in food packaging industries. In the present work, we have developed a new food wrap method by cross-linking cinnamaldehyde on low-density polyethylene (Cin-C-LDPE) using novel photocrosslinker technology. The cinnamaldehyde showed potent antimicrobial activity (145mg/mL of MIC) against MRSA 090 and radical scavenging activity (RSA). The cinnamaldehyde was successfully cross-linked to LDPE and exhibited excellent antibiofilm properties against MRSA090 compared to bare LDPE. This positive interaction of developed Cin-C-LDPE against MRSA090 biofilm was confirmed by SEM and FT-IR studies, and results showed the damaged cell membrane architecture, inturn abridged adherence of MRSA090. The Cin-C-LDPE wrapped chicken, mutton, cheese, and grapes showed 2.5±0.15 log MRSA 090 reduction at the end of 10th day compared to the bare LDPE wrapped food samples. This clearly concludes that for the first time we have developed a novel Cin-C-LDPE food wrap technology effectively involved in biocidal activity against MRSA090. Applying this new strategy to develop food wrap containing nontoxic natural antimicrobial to target cell membrane components is the upcoming challenging and promising research gap remains in the food packaging industry for the future world.

MALDI-TOF-MS based identification and molecular characterization of food associated methicillin-resistant Staphylococcus aureus.

Food-borne methicillin resistant Staphylococcus aureus (MRSA) is involved in two-fold higher mortality rate compared to methicillin susceptible S. aureus (MSSA). Eventhough Mysuru recognized as cleanest city in the world, prevalence of food contamination is not detailed. The aim is to screen food samples from Mysuru area and to characterize MRSA strain, employing MALDI-Biotyper, multiplex PCR to distinguish between MRSA and MSSA by PCR-coupled single strand conformation polymorphism (PCR-SSCP). Of all the food-borne pathogens, S. aureus contamination accounts for 94.37 ± 0.02% (P < 0.01), strains characterized by means of nuc genes, followed by species specific identification by Coa, Eap and SpA genes and multiplex PCR to confirm the presence of three methicillin resistant staphylococcal species simultaneously using nuc and phoP genes. Amplification of mecA gene in 159 isolates confirmed that all strains are methicillin resistant, except UOM160 (MSSA) and multi-drug resistant (MDR) in 159 isolates confirmed by 22 sets of ß-lactam antibiotics. MSSA and MDR-MRSA were discriminated by PCR-SSCP using nuc gene for the first time. From the present studies, compared to conventional methods MALDI-Biotyper emerged as an effective, sensitive (>99%), robust (<2 min), and alternative tool for pathogen identification, and we developed a PCR-SSCP technique for rapid detection of MSSA and MRSA strains.

Promising biocidal activity of thymol loaded chitosan silver nanoparticles (T-C@AgNPs) as anti-infective agents against perilous pathogens.

The advent of biodegradable polymer-encapsulated drug nanoparticles has made an exciting area of drug delivery research. The present study investigated novel and simple route for synthesis of thymol loaded chitosan silver nanoparticles (T-C@AgNPs) using chitosan and thymol as reducing, capping agent respectively to understand the therapeutic efficacy. The UV-vis spectroscopy, DLS, FT-IR, SEM, EDS, XRD used for characterization and radical scavenging activity, anti-microbial and biocompatibility was taken to ascertain an efficacy of novel T-C@AgNPs. The T-C@AgNPs intense peak at 490nm indicates the formation of nanoparticles and had average particle size of 28.94nm with spherical shape, monodisperse state in water, also exhibited excellent biocompatibility of cubic shaped pure silver element containing T-C@AgNPs. The antibacterial activity was studied for gram positive and gram negative food-borne pathogens and effective inhibition at 100µgmL-1 to S. aureus, S. epidermidis, S. haemolyticus (10.08, 10.00, 11.23mm) and S. typhimurium, P. aeruginosa and S. flexneri (9.28, 9.33, 12.03mm) compared to antibiotic Streptomycin. This study revealed the efficacy against multiple food-borne pathogens and therapeutic efficacy of T-C@AgNPs offers a valuable contribution in the area of nanotechnology. This proved to be a first-class novel antimicrobial material for the first time in this study.

Novel T-C@AgNPs mediated biocidal mechanism against biofilm associated methicillin-resistant Staphylococcus aureus (Bap-MRSA) 090, cytotoxicity and its molecular docking studies.

Staphylococcus aureus is a commonly found pathogen that can cause food-spoilage and life threatening infections. However, the potential molecular effects of natural active thymol molecules and chitosan silver nanoparticles (C@AgNPs) in bacteria remain unclear. This gap in the literature has prompted us to study the effects of thymol loaded chitosan silver nanoparticles (T-C@AgNPs) against biofilm associated proteins in methicillin-resistant S. aureus (Bap-MRSA) 090 and also their toxicity, anti-cancer activity, and validation of their in silico molecular docking. The results showed excellent antibacterial activity of T-C@AgNPs against Bap-MRSA 090, having a minimum inhibitory concentration of 100 µg mL-1 and a 10.08 ± 0.06 mm zone of inhibition (ZOI). The cyclic voltammogram (CV) analysis clearly showed pore forming of T-C@AgNPs at 300 µg mL-1 concentration, and evidence of the interruption of the electron transport chain was clearly seen. The 200 µg mL-1 concentration exhibited a 52.60 ± 0.25% anti-biofilm property by T-C@AgNPs against Bap-MRSA 090. The T-C@AgNPs showed no toxicity to peripheral blood mononuclear cells (PBMC) (IC50 = 221 ± 0.71 µg mL-1) compared to the control, and anti-cancer activity against human triple negative breast cancer cell line (MDA-MB-231) (IC50 110 ± 1.0 µg mL-1) compared to the standard drug Doxorubicin (IC50 = 19 ± 1.0). The excellent properties of T-C@AgNPs were validated by in silico molecular docking studies and showed best match scoring to target proteins compared to standards. These excellent properties of T-C@AgNPs highlight for the first time its pharmacology and potential in medicinal drug development applications for future research.

A rapid method for isolation of genomic DNA from food-borne fungal pathogens.

Food contaminated with fungal pathogens can cause extremely harmful effects to human even when present in low concentrations. Researchers now pay more attention towards rapid DNA extraction for the quick screening, which is highly demanded in diverse research field. Molecular description of many fungal species is identified by different molecular characteristics. Hence, the efficient isolation of genomic DNA and amplification using PCR is a prerequisite for molecular characterization. Here, we used an improved Sodium dodecyl sulfate-Cetyltrimethyl ammonium bromide-Chloroform-isoamyl alcohol method by combining Sodium dodecyl sulfate with cetyl methylammonium bromide without addition of proteinase K, RNase K, and ß-mercaptoethanol. To analyze the quality of recovered DNA, this method was compared with the other four routine methods. The present method has been chosen in the study as a preferred method because of easy adaptation to routine laboratories/food industries considering its rapid, sensitivit,y and cost effectiveness involved in the method.

Synthesis and antimicrobial study of novel heterocyclic compounds from hydroxybenzophenones.

The triazolothiadiazine analogues 6a-e were obtained via a multistep synthesis sequences beginning with the hydroxybenzophenones 1a-e. Hydroxybenzophenones on reaction with ethyl chloroacetate affords ethyl (2-aroylaryloxy)acetates 2a-e which on treatment with hydrazine hydrate yields 2-(2-aroylaryloxy)acetohydrazides 3a-e. Intramolecular cyclization of 3a-e with carbon disulfide affords 5-(2-aroylaryloxy)methyl-1,3,4-oxadiazole-2-(3H)thiones 4a-e, which on treatment with hydrazine hydrate yields 4-amino-5-(2-aroyl aryloxy)methyl-1,2,4-triazole-3-(2H)thiones 5a-e. Condensation of 5a-e with alpha-halocarbonyl compound results in 3-(2-aroylaryloxy)methyl-6-phenyl-1,2,4-triazolo[3,4-b][1,3,4] thiadiazine 6a-e analogues. The compounds 4a-e, 5a-e and 6a-e were tested against variety of fungal and bacterial strains in comparison to fluconazole and chloramphenicol, respectively.

Multiplex PCR for simultaneous identification of Ralstonia solanacearum and Xanthomonas perforans.

Ralstonia solanacearum is a causative agent of bacterial wilt in many economically important crops, and Xanthomonas perforans is the causal organism of bacterial spot, one of the most important diseases of vegetables. A multiplex PCR protocol has been developed for the simultaneous, specific and rapid identification of R. solanacearum and X. perforans in plant materials. Species-specific primers RS-F-759 and RS-R-760 for R. solanacearum, RST2 and RST3 for X. perforans were used for identification of both pathogens at primer concentrations of 1:4 by optimization of multiplex PCR at annealing temperature of about 61 ± 1 °C. With these primer sets, specific amplification of 281- and 840-bp PCR products was obtained for R. solanacearum and X. perforans, respectively. The multiplex PCR assay was validated with susceptible plants mechanically inoculated with both the pathogens; specific PCR products confirmed the presence of R. solanacearum and X. perforans. The multiplex PCR is valuable in identification as well as primary screening of cultivars of both pathogens. The present study is a rapid and easy method for early identification of pathogens from asymptomatic and symptomatic plant materials.

Identification of genes associated with black rot resistance in cabbage through suppression subtractive hybridization.

The suppression subtractive hybridization was employed to elucidate the resistance mechanism in Brassica oleracea var. capitata upon infection with Xanthomonas campestris pv. campestris. A cDNA library was constructed enriched in differentially expressed transcripts in the resistant cultivar Pusa mukta. A total of 150 unigenes were classified into five functional categories. The present study indicates that the defense-related unigenes accounted for the 35 % of the total unigenes studied. Confirmation of defense-specific representation genes through semiquantitative RT-PCR revealed their increased expression in the resistant cultivar which was validated by qPCR. The resistant cultivar elicited a strong hypersensitive response upon attack by black rot pathogen. The study is first of its kind where the resistant cultivar Pusa mukta in India has been assessed for its resistance to the bacterial pathogen.

Vegetable oil blends with α-linolenic acid rich Garden cress oil modulate lipid metabolism in experimental rats.

Vegetable oil blends with modified fatty acid profile are being developed to improve n-6/n-3 polyunsaturated fatty acid (PUFAs) ratio in edible oils. The objective of this study is to develop vegetable oil blends with α-linolenic acid (ALA) rich Garden cress oil (GCO) and assess their modulatory effect on lipid metabolism. Sunflower oil (SFO), Rice bran oil (RBO), Sesame oil (SESO) were blended with GCO at different ratios to obtain n-6/n-3 PUFA ratio of 2.3-2.6. Native and GCO blended oils were fed to Wistar rats at 10% level in the diet for 60 days. Serum and liver lipids showed significant decrease in Total cholesterol (TC), Triglyceride (TG), LDL-C levels in GCO and GCO blended oil fed rats compared to native oil fed rats. ALA, EPA, DHA contents were significantly increased while linoleic acid (LA), arachidonic acid (AA) levels decreased in different tissues of GCO and GCO blended oils fed rats. In conclusion, blending of vegetable oils with GCO increases ALA, decreases n-6 to n-3 PUFA ratio and beneficially modulates lipid profile.

Lytic activity in pearl millet: its role in downy mildew disease resistance.

Sclerospora graminicola causes downy mildew disease in susceptible pearl millet. Molecular basis of downy mildew disease resistance has been studied. Coleoptile region has been shown earlier to be the most susceptible site for attack by the pathogen. Lytic activity is differentially expressed in the coleoptile region of 3-day-old pearl millet seedlings of resistant and susceptible cultivars. Significantly higher levels of lytic factors were measured in the coleoptile region of resistant cultivars (100%) than in that of susceptible cultivars (20%). Both constitutive and inducible lytic factors were observed in different resistant cultivars, and they were able to lyse the pathogen. The level of lytic activity correlated well with the degree of resistance as evaluated by field screening studies. The present study, therefore, proposes that lytic factors found in the coleoptile region of the pearl millet seedling, are responsible for the lysis of the pathogen in the resistant plant, and may therefore provide resistance to downy mildew disease. This study also provides a simple method to evaluate downy mildew resistance in pearl millet cultivars.